This invention relates to a method of performing ligase chain reaction (LCR) amplification and, particularly, to a method of ligase chain reaction amplification wherein at least one of the probes is reversibly modified at the intended point of ligation so that it is not a substrate for the ligase catalyzed reaction. Exemplary modifications include the absence of one or more nucleic acid bases to form a "recess". The modified end prevents or reduces target independent blunt-end ligation of probe duplexes and is later corrected in a target dependent manner to enable ligation.
In many cases, the feasibility of a nucleic acid based diagnostic assay is dependent on the ability to amplify the signal generated by only a few molecules of target. Although signal amplification is one potential solution, target amplification is often the preferred solution in nucleic acid based assays. Target amplification involves the repeated copying or duplication of sections of the nucleic acid designated as the target.
In the target amplification technique known as polymerase chain reaction (PCR) a pair of primers are employed in excess to hybridize at the outside ends of complementary strands of the target nucleic acid. The primers are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences themselves, following dissociation from the original target strand. New primers are then hybridized and extended by a polymerase, and the cycle is repeated to increase geometrically the number of target sequence molecules. PCR is described further in U.S. Pat. Nos. 4,683,195 and 4,683,202.
An alternate mechanism for target amplification is known as ligase chain reaction (LCR). In LCR, two primary (first and second probes) and two secondary (third and fourth) probes are employed in excess. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate-3' hydroxyl relationship and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes will also hybridize to the target complement in the first instance. Once the fused strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary fused product. In order to understand LCR and the improvements described herein, it is important to realize that the fused products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described more completely in EP-A-320 308, the entire disclosure of which is incorporated herein by reference.
A potential problem associated with ligase chain reaction is background signal caused by target independent ligation of the probes. Since the third probe hybridizes to the first probe and the fourth probe hybridizes to the second probe, the probes, which are added in excess, can easily form duplexes among themselves. These duplexes can become ligated independently of the presence of target to form a fused product which is then indistinguishable from the desired amplified target, yet which is still capable of supporting further amplification. Although target independent blunt-end ligation of these duplexes is a relatively rare event, it is sufficiently common to cause undesirably high background signals in diagnostic assays.
Some attempts to overcome this background problem have been published. For example, WO 90/01069 (Segev Diagnostics) and GB 2 225 112 A (Imperial Chemical Industries Plc.) describe versions of a ligation-based amplification scheme which includes a polymerase-mediated gap-filling step prior to ligation. However, these references teach nothing about selection of the particular target sequences as taught and claimed in the present application.
It is therefore a primary object of the present invention to improve the sensitivity of nucleic acid based assays by decreasing the occurrence of target independent ligation which causes falsely positive background signal. This object is met in the present invention by modifying at least one probe end so that when hybridized with its complementary probe, the resulting duplex is not "blunt-ended" (i.e. ligatable) with respect to the partner complementary probe duplexes. After hybridizing to the target, the modified ends are "corrected" in a target dependent fashion to render the adjacent probes ligatable. Several features of the probes and the associated target sequences taught in this application make this task particularly elegant.
According to one feature, the probes have recesses relative to the point of ligation which create a gap when hybridized to the target. The gap is then filled in a target dependent manner to render the probes ligatable. Gap filling can be accomplished by extension of one or more probes, preferably two probes. Targets are selected and probes are designed so that only one, or a maximum of two, of the four deoxyribonucleotide triphosphates is needed to fill both the gaps.